Recently, there has been a growing concern on the necessity of a periodic checkup on the blood glucose level for the diagnosis and prevention of diabetes. The blood glucose level can be easily measured using a portable blood glucose monitoring device, in particular, using a strip type biosensor by each individual.
However, it is inconvenient to use because the results of blood glucose measurements varies depending on whether a patient has taken a meal or not, and the measured value may vary depending on the patient's physical conditions on the examination day, e.g., stress, drinking, fatigue, etc.
In contrast, when a diabetic patient is measured and managed using glycated hemoglobin (HbA1c), the result is not affected by factors such as physical conditions of the patient or whether the patient has taken a meal or not. It is generally accepted that HbA1c value reflects the average blood glucose level for a period of about 3 to 4 months, and thus can be used as an index for evaluating the efficiency of the patient's method of managing the blood glucose level. This is because the glucose present in blood reacts with hemoglobin, the blood constituting protein, for a long period of time, undergoes the Amadori rearrangement, and forms a modified protein of hemoglobin-glucose, the so-called glycated hemoglobin (HbA1c). The structure of the HbA1c is shown below.

Since the life cycle of the thus formed HbA1c is about from 90 to 120 days, it can provide a very important data for the diagnosis of diabetes of a patient or the efficiency of blood glucose management by the patient based on the average blood glucose level.
The clinical evaluation of the HbA1c is measured by the HbA1c's relative ratio to the total hemoglobin concentration in terms of percentage. The HbA1c value of 6.0% or less is considered normal. The HbA1c value is a very important factor in managing diabetes complications. According to reports, for example, when the HbA1c value is lowered by 1% it has the effects of reducing the risk of myocardial infarction by 14%, cataract by 19%, microangiopathy by 37%, peripheral arterial disease by 43%, death from diabetes by 21%, etc.
Although routine measurement of a patient's blood glucose level is important to check the patient's daily health conditions and provide an appropriate treatment for the patient accordingly, it is still necessary to measure the patient's glycated protein or HbA1c level in order to determine the patient's long-term blood glucose management status, prevent complications, and provide a suitable treatment for the patient.
A standard method of measuring the HbA1c level in blood, which serves as an important index in blood glucose management, may include hemolyzing a collected blood sample by mixing it with a suitable reagent, e.g., a surfactant such as TX-100, passing it through an HPLC ion-exchange or affinity column packed with beads/gels with a functional surface of boronic acid derivatives, measuring particular wavelength of proteins separated therefrom via visible light apart from normal proteins, thereby calculating the ratio of glycated proteins.
However, the standard method described above has a disadvantage that it can be only performed in a well-facilitated major clinical laboratory or a hospital's central laboratory by a trained clinical professional and the operation and maintenance is relatively costly, esp. when fewer tests are ordered. Additionally, the blood HbA1c level may be measured using HbA1c antibodies. However, the amount of the supplies is very limited due to the lack of companies producing the same, and also the apparatus is very complicated requiring a reaction-separation step.
U.S. Pat. No. 5,541,117 discloses a method for determining hemoglobin content via reflectance photometry after preparing a pad on which antibodies are coupled and allowing a blood sample to be hemolyzed and introduced onto the pad. However, its drawbacks include using high cost antibodies, and having a difficulty in manufacturing sensors with uniform quality due to non-uniformity in porous pads. Additionally, the relative amount of the glycated protein is determined electrochemically using a marker compound after separating proteins in the sample via an antibody modified solid phase. However, glycated proteins and glycated protein-marker complex competitively gather around the surface of electrodes and signals from the electrochemical reactions between the marker and injected substrate is largely inhibited. Therefore the measurement of glycated proteins is compromised and performance with regard to reproducibility is undermined.
As an alternative, an enzymatic method may be used to measure the concentration of a glycated protein such as HbAlc. The enzymatic method includes introducing a protease to the glycated protein of a given sample, performing a pretreatment to release the glycated peptides or glycated amino acids, which are used as a substrate for the following reaction, where an intermediate product (e.g., hydrogen peroxide) is produced by acting a glycated peptide-specific enzyme or glycated amino acid-specific enzyme (e.g., fructosyl peptide oxidase or fructosyl amino acid oxidase) on the separated substrate, and followed by a signal-producing peroxidase-hydrogen peroxide reaction with an appropriate color developing agent, e.g. a detectable product.
Japanese Patent Application Publication No. H5-192193 discloses a method for a specific quantification of fructosamine by specifically recognizing the ketoamine structure present in fructosamine, separating ketoamine oxydase, which is a novel enzyme catalyzing the oxidation of the ketoamine structure, from various microorganisms, oxidizing only the fructosamine pretreated with a protease in the presence of the ketoamineoxidase, and measuring the resulting product, i.e., glucosone or hydrogen peroxide.
Japanese Patent Application Publication No. 2001-95598 discloses a method for measuring hydrogen peroxide formed by treating a glycated protein-containing sample with a protease, separating a glycated peptide, preferably an α-glycated peptide, more preferably an α-glycated dipeptide, from the glycated protein, and then acting oxydase on the separated glycated peptide, or a method of measuring the separated glycated peptide via HPLC, and also discloses a method of measuring the glycated peptide in a given sample, and a reagent kit for the measurement via an enzymatic method.
HbAlc is a compound in which glucose is bound to a valine residue of N-terminal β chain of hemoglobin A (HbA) (α2β2), and the degree of glycation is indicated in terms of percentage (%) or mmol of HbAlc concentration relative to the total hemoglobin concentration (mole). According to the enzymatic method of the present invention, N-terminal β chain of hemoglobin to which glucose is bound should be converted into monomeric substances of fructocyl amino acid by a protein-digesting protease. The thus formed fructocyl amino acid reacts with fructocyl peptide oxidase (FPOX), an oxidase, to generate hydrogen peroxide, and the hydrogen peroxide generated thereof is oxidized by peroxidase (POD), and the concentration of the HbAlc is measured by analyzing the degree of color development of a substrate through a reduction caused by electrons released by the oxidation via a spectrophotometric analysis.
The enzymatic method using various enzymes as described above has an advantage that it can provide a more accurate result due to the enzyme's characteristic of selectivity compared to other methods, but it has a disadvantage that it requires a long reaction time and its reaction sensitivity is low due to sophisticated reaction conditions.
The inventors of the present invention, while endeavoring to find a method to improve reaction rate and reaction sensitivity in the enzymatic method for the quantification of HbAlc, discovered that when a Zwitterionic surfactant and a nitrous compound containing reagent composition is added to a hemolysis reagent, it considerably accelerates the hemolysis rate and also induces the N-terminal β-chain of hemoglobin released by hemolysis to be exposed to the outside of the hemoglobin molecule and assists the participation of a greater number of molecules thereby improving reaction sensitivity and reaction rate of the HbAlc, and the nitrous compound makes the protein structure of the hemoglobin flexible so that the amino acid sequence of the N-terminal β-chain of hemoglobin can be readily cleaved off, thereby significantly reducing the entire time required for the measurement and improving the accuracy of the measurement, thus completing the present invention.